Heparin, a polydisperse mucopolysaccharide, is the most commonly used clinical anticoaguant. Despite the use of over 50 million yearly doses in the U.S. alone, heparin's exact chemical structure and the precise nature of it's anticoagulant activity are still unknown. Heparin's primary biological activity is as an anticoagulant. It is believed to act by binding factors in the coagulation cascade like Antithrombin at specific sequences in it's chain. "Heparin is the drug responsible for most deaths in otherwise healthy patients", according to the Boston Collaborative Drug Surveillance Program. Heparin anticoagulation results in hemorraghe in 8-33 percent of patients on heparin therapy. Heparin's anticoagulant activity is difficult to separate from it's secondary activities which cause a myriad of side effects: some beneficial like decreased atherosclerosis and some harmful like thrombocytopenia. Heparin is neither orally active nor is it effectively utilized when given subcutaneously, and due to it's polydispersity when administered intra venously it is difficult to predict it's half life. I propose research towards four specific aims: 1) the development of methodology for heparin structure determination applicable to the determination of complex polysaccharides; 2) the determination of heparin's structure; 3) the elucidation of the relationship of heparidin's structure to it's activity; 4) the preparation and testing of new heparin-derived anticoagulants. Two major tools will be used to determine heparin's structure. First, a microbial heparinase will be utilized to take heparin apart in a predictable, defined fashion resulting in smaller more manageable fragments. Second, a computer will establish the sequence in which these fragments were originally arraigned. Three different sequencing strategies will be used depending on whether heparin is determined to have a random, semi-random, or defined sequence. The methodology will be general so as to be applicable to the sequencing of other polysaccharides. To relate heparin's structure to it's activity, heparin fragments will be fractionated by their biological activities using affinity chromatography. Promising fragments with high, or differential activities in vitro will be tested for efficacy and acute toxicity in vivo. In preliminary studies we prepared heparinase derived fragements, as small as tetrasaccharides, which inhibit one coagulation factor but not a second. Additionally, such small fragments dialyze, are orally active, may have enhanced subcutaneous activity, and because they are pure defined subtances their half-lives in vivo may be more predictable.